- Email: [email protected]
EFFECTS OF CRYOPROTECTANTS ON HUMAN OOCYTE
632
significant and consistent effect this drug should not be used to treat patients with either oral or genital herpes. Academic Department of Genito-Urinary Medicine, Middlesex Hospital Medical School,
A. MINDEL
London W1N8AA 1. Wickett WH, Bradshaw
LJ, Wilson J, Glasky AJ. Clinical effectiveness of the immunopotentiating agent, inosiplex, in herpes virus infections. Proc 76th Meet Am Soc
Microbiol 1976; 260: 211. 2. Lassus A, Salo OP. Isoprinosine versus placebo in the treatment of recurrent genital herpes. Proc 1st Wld Congr Sex Trans Dis (Puerto Rico, 1981). 3. Bouffaut P, Saurat JH. Isoprinosine as a therapeutic agent in recurrent mucocutaneous infections due to herpes virus. Int J Immunopharmacol 1980; 2: abstr. 4. Galli M, Lazzarin A, Moroni M, Zanussi C. Isoniplex in recurrent herpes simplex infections. Lancet 1982; ii: 331. 5. Corey L, Chiang WT, Reeves WC, Stamm WE, Brewer L, Holmes KK. Effect of isoprinosine on the cellular immune response in initial genital herpes virus infections. Clin Res 1979; 27: 41A (abstr). 6. Nilsen AE, Aasen R, Halsos AM, et al. Efficacy of oral acyclovir in the treatment of initial and recurrent genital herpes. Lancet 1982; ii: 571-73. 7 Bryson YJ, Dillon M, Lovett M, et al. Treatment of first episodes of genital herpes simplex virus infection with oral acyclovir. N Engl J Med 1983; 1: 95-98. 8. Mertz GJ, Critchlow CW, Benedetti J, et al. Double blind placebo controlled trial of oral acyclovir in first episode genital herpes simplex virus infection. JAMA 1984; 252: 1147-51.
Fig 1-Dimensional changes
of human oocytes
during addition and
removal of glycerol.
LASER-ASSISTED VASCULAR ANASTOMOSIS
SiR,-Dr Quigley and his colleagues (Feb 9, p 334), in their letter laser-assisted vascular anastomosis, criticise my contribution to
PB1=modified Dulbecco’s phosphate.
on
correspondence columns (Oct 6, p 816). My letter was a brief description of the clinical applications of the technique of microvascular anastomosis with the neodymium-YAG laser. The details are given elsewhere. 1-3 To the best of my knowledge, based on my last visit to Northwestern University Medical School in May, 1984, my neodymium-YAG laser technique has not been repeated by Quigley or any of his co-workers. They do not have the micromanipulator described by me and the spot size of 4 mm they mention is ten times bigger than the 0 -3mm spot size I use, and this
your
difference invalidates any
comments
of
Quigley
et
al
on
my
technique.
Quigley et al are interested in a particular CO2 milliwatt laser and do not mention the fact that the vascular anastomosis achieved by them is not sutureless, but only supplements the conventional suture technique. I have not been able to find any properly documented publication on the use of the milliwatt CO2 laser in vascular anastomosis. Quigley et al are "certain that basic mechanisms of tissue welding is the same for all lasers". This is ridiculous: in the first place, the basic mechanism for laser tissue welding is not fully understood and, secondly, differences in the biological effects of lasers of different wavelengths are well accepted. This is why milliwatt CO2 lasers achieve poorer results in vascular anastomosis. Those who study my technique and apply it properly will not have any of the problems and doubts Quigley et al mention. Since my technique is not yet fully automated, microsurgical skill is still required for a successful outcome. My technique should not be attempted without proper equipment and training in the safe use of it. Misuse of the 4 neodymium-YAG laser can lead to
cryopreservation, confirms that human embryos can be successfully recovered after storage at -196°C. Cryopreservation of embryos raises many ethical problems, and it may be more acceptable to freeze unfertilised oocytes. With this in mind we have been studying the cryopreservation of human preovulatory oocytes. With low concentrations of glycerol as cryoprotectant and normothermic equilibration temperatures mouse oocytes can be successfully cryopreserved with subsequent culture to expanded blastocysts after thawing and in-vitro fertilisation.2 Using a similar protocol, we soon found that human oocytes required at least 45 min equilibration time in glycerol with an increase in glycerol removal time (fig 1). After removal of cryoprotectant freshly produced sperm was added to media containing oocytes in an attempt at fertilisation. Fertilisation was not observed in any of the oocytes so treated. High survival rates of eight-cell mouse embryos is possible after freezing with 1,2-propanediol as cryoprotectant and an
complications.
Walker Institute, 1964 Westwood Blvd, Suite 220, Los Angeles, California 90025, USA
K. K. JAIN
Jain KK. Sutureless microvascular anastomosis using a Nd:YAG laser. J Microsurg 1980; 1: 436-39. 2. Jam KK. Sutureless end-to-side microvascular anastomosis using Nd:YAG laser. Vasc 1.
Surg 1983; 17:
240-43.
3.
Jam KK. Handbook of laser neurosurgery. Springfield, Illinois: Charles C Thomas,
4.
Jain KK. Complications of use of Nd:YAG press).
1983. laser in neurosurgery.
Neurosurgery (in
EFFECTS OF CRYOPROTECTANTS ON HUMAN OOCYTE
SiR,-The report from Australial of a pregnancy with subsequent live birth after transfer of an eight-cell human embryo, after
Fig 2-Dimensional changes of human oocytes during addition and removal of 1,2-propanediol.
633 time of only 15 min.3When we submitted human oocytes to this regimen rapid shrinkage of the blastomere was observed followed by an equally swift expansion during equilibration. After 10 min immersion in 1,2-propanediol, oocytes had recovered to 90% of their original volume (fig 2). Removal of 1,2-propanediol was equally rapid. After the addition of sperm regular cleavage was observed, and at the eight-cell stage the experiment was terminated. With the increase in demand for in-vitro fertilisation (IVF) and embryo transfer and the low success rates so far, it would seem to be more prudent to store excess oocytes for transfer in subsequent cycles, perhaps in the more favourable hormonal environment of a natural cycle. Such a technique could be beneficial in the treatment of IVF patients where several oocytes are recovered, but clearly much work is needed before a protocol for the successful storage and recovery of human oocytes can be devised.
equilibration
TABLE I-EFFECT OF INTRAPERITONEAL IMIPENEM ON GUINEAPIGS INFECTED WITH L PNEUMOPHILA
Imipenem given means
over a
period of 5 days beginning 1 day post infection. Temperatures day 4 controls (two) and 20 mglday group (three).
of six values except for
TABLE II-NUMBERS OF L PNEUMOPHILA IN GUINEAPIG LUNGS AFTER
Supported in part by the Special Trustees of the Royal Free Hospital. A. BERNARD D. A. IMOEDEMHE R. W. SHAW B. FULLER
Academic
Departments of Obstetrics Gynaecology and Surgery, Royal Free Hospital School of Medicine, London NW3 2QG 1. Trounson
A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight cell embryo. Nature 1983, 305: 707. 2. Fuller BJ, Bernard A. Successful in vitro fertilization of mouse oocytes after cryopreservation using glycerol. Cryo-Letters 1984; 5: 307-12. 3. Renard JP, Babmet C. High survival of mouse embryos after rapid freezing and thawing inside plastic straws with 1,2-propanediol as cryoprotectant. J Exp Zoo 1984; 230: 443-48.
removed and diluted in distilled water. L pneumophila was then assayed by growth on BCYE agar. L pneumophila did not multiply in tissue culture medium and 0 8% digitonin did not affect the viability of the organism over 2 h. Imipenem, although not preventing death, did marginally reduce pyrexia and prolonged the average survival time, all deaths occurring within the period of therapy (table i). Lungs from guineapigs treated with imipenem contained marginally fewer L pneumophila than those from untreated infected controls (table n). Lesions in the imipenem-treated and untreated infected control groups were identical and typical of legionnaires’ disease.7 Whereas erythromycin, an antibiotic currently recommended for treatment of legionnaires’ disease, was bactericidal against intracellular L pneumophila at concentrations approximating to the MIC (0 -05 g/ml), imipenem did not affect intracellular survival at concentrations 200 times higher than its MIC (table III). Thus imipenem had no effect on lung lesions or in preventing death in guineapigs infected with L pneumophila aerosol but it was slightly beneficial in reducing pyrexia and lung infection and in extending survival time. This was probably a result of bactericidal action on extracellular organisms. In our animal model, by contrast, erythromycin and some other antibiotics eliminated L pneumophila from the lung and prevented death.9 Imipenem may reduce bacwas
EFFICACY OF IMIPENEM IN EXPERIMENTAL LEGIONNAIRES’ DISEASE
SIR,-Imipenem has been used successfully in two patients with legionnaires’ disease,’ but this drug did not prevent the death of2 guineapigs infected intratracheally with Legionella pneumophila.2 Although imipenem has a favourably low minimum inhibitory concentration (MIC) against L pneumophila (0 - 06 J4g/ml) the main factor in estimating its efficacy against this intracellular opportunistic pathogen is thought to be its penetration into phagocytes.l,2 We have evaluated the efficacy of imipenem in guineapigs infected with a small particle aerosol containing L pneumophila3and have assessed the penetration of imipenem into alveolar phagocytes infected in vivo. L pneumophila (Corby strain, serogroup 1) was grown for 4 days at 37°C on BCYE agar4and resuspended in distilled water, inocula being standardised from the total count. Respiratory infection was achieved via a Henderson apparatus in conjunction with a three-jet Colison spray to generate bacterial aerosols containing particles <5 pm in diameter at an optimum relative humidity of65%.3 The animals studied were female Dunkin-Hartley guineapigs of category 4 health statusS weighing about 330 g. Imipenem (donated by Merck Sharp and Dohme) was given intraperitoneally as 5 or 10 mg doses every 6 h over 5 days, this dose being roughly equivalent to 4 or 8 g daily in man. Average survival times were calculated, after exclusion of survivors to the 10th day or beyond, by dividing the total number of days animals survived before dying by the total number of animals which died. To obtain infected alveolar guineapigs were killed about 6 h after aerosol infection and the lungs were washed by instillation and withdrawal of 3 x 10 ml of minimum essential medium (MEM) containing sodium bicarbonate (10 mmol/1), "hepes" buffer (20 mmol/1), L-glutamine (2 mmol/1), and heparin (5 U/ml). The washings were centrifuged at 200 g for 10 min and resuspended in fresh medium containing 5% v/v normal autologous guineapig serum. Cells were standardised at 106 /ml and 0-2 ml volumes were placed in the wells of a plastic tissue culture plate and incubated for about 3 h at 37°C in 5% CO2 in air. The cells were then washed with fresh medium (0.22 ml x 2) and incubated in 0 - 2 ml of MEM containing 5% autologous guineapig serum plus antibiotics at 37°C for 24 and 48 h in 5% CO2 in air. At 0, 24, and 48 h the monolayer of cells in each well was gently washed twice with 0 - 2 ml of fresh medium, and 0 - 2 ml 0 - 8% digitonin in distilled water was added. When the cells had disintegrated the suspension
teraemia and consequent dissemination of L pneumophila but, as our studies show, this drug does not penetrate alveolar phagocytic cells TABLE III-EFFECT OF IMIPENEM ON THE REPLICATION OF L PNEUMOPHILA WITHIN GUINEAPIG ALVEOLAR PHAGOCYTES
phagocytes
Figures in body of table denote log (viable organisms per well) and are the mean of2 values. Deviation between values was <0’3 3 log. Changmg imipenem or erythromycin at 6 or 8 hourly intervals respectively in a parallel series of wells did not alter the counts at 24 h.
,
significant and consistent effect this drug should not be used to treat patients with either oral or genital herpes. Academic Department of Genito-Urinary Medicine, Middlesex Hospital Medical School,
A. MINDEL
London W1N8AA 1. Wickett WH, Bradshaw
LJ, Wilson J, Glasky AJ. Clinical effectiveness of the immunopotentiating agent, inosiplex, in herpes virus infections. Proc 76th Meet Am Soc
Microbiol 1976; 260: 211. 2. Lassus A, Salo OP. Isoprinosine versus placebo in the treatment of recurrent genital herpes. Proc 1st Wld Congr Sex Trans Dis (Puerto Rico, 1981). 3. Bouffaut P, Saurat JH. Isoprinosine as a therapeutic agent in recurrent mucocutaneous infections due to herpes virus. Int J Immunopharmacol 1980; 2: abstr. 4. Galli M, Lazzarin A, Moroni M, Zanussi C. Isoniplex in recurrent herpes simplex infections. Lancet 1982; ii: 331. 5. Corey L, Chiang WT, Reeves WC, Stamm WE, Brewer L, Holmes KK. Effect of isoprinosine on the cellular immune response in initial genital herpes virus infections. Clin Res 1979; 27: 41A (abstr). 6. Nilsen AE, Aasen R, Halsos AM, et al. Efficacy of oral acyclovir in the treatment of initial and recurrent genital herpes. Lancet 1982; ii: 571-73. 7 Bryson YJ, Dillon M, Lovett M, et al. Treatment of first episodes of genital herpes simplex virus infection with oral acyclovir. N Engl J Med 1983; 1: 95-98. 8. Mertz GJ, Critchlow CW, Benedetti J, et al. Double blind placebo controlled trial of oral acyclovir in first episode genital herpes simplex virus infection. JAMA 1984; 252: 1147-51.
Fig 1-Dimensional changes
of human oocytes
during addition and
removal of glycerol.
LASER-ASSISTED VASCULAR ANASTOMOSIS
SiR,-Dr Quigley and his colleagues (Feb 9, p 334), in their letter laser-assisted vascular anastomosis, criticise my contribution to
PB1=modified Dulbecco’s phosphate.
on
correspondence columns (Oct 6, p 816). My letter was a brief description of the clinical applications of the technique of microvascular anastomosis with the neodymium-YAG laser. The details are given elsewhere. 1-3 To the best of my knowledge, based on my last visit to Northwestern University Medical School in May, 1984, my neodymium-YAG laser technique has not been repeated by Quigley or any of his co-workers. They do not have the micromanipulator described by me and the spot size of 4 mm they mention is ten times bigger than the 0 -3mm spot size I use, and this
your
difference invalidates any
comments
of
Quigley
et
al
on
my
technique.
Quigley et al are interested in a particular CO2 milliwatt laser and do not mention the fact that the vascular anastomosis achieved by them is not sutureless, but only supplements the conventional suture technique. I have not been able to find any properly documented publication on the use of the milliwatt CO2 laser in vascular anastomosis. Quigley et al are "certain that basic mechanisms of tissue welding is the same for all lasers". This is ridiculous: in the first place, the basic mechanism for laser tissue welding is not fully understood and, secondly, differences in the biological effects of lasers of different wavelengths are well accepted. This is why milliwatt CO2 lasers achieve poorer results in vascular anastomosis. Those who study my technique and apply it properly will not have any of the problems and doubts Quigley et al mention. Since my technique is not yet fully automated, microsurgical skill is still required for a successful outcome. My technique should not be attempted without proper equipment and training in the safe use of it. Misuse of the 4 neodymium-YAG laser can lead to
cryopreservation, confirms that human embryos can be successfully recovered after storage at -196°C. Cryopreservation of embryos raises many ethical problems, and it may be more acceptable to freeze unfertilised oocytes. With this in mind we have been studying the cryopreservation of human preovulatory oocytes. With low concentrations of glycerol as cryoprotectant and normothermic equilibration temperatures mouse oocytes can be successfully cryopreserved with subsequent culture to expanded blastocysts after thawing and in-vitro fertilisation.2 Using a similar protocol, we soon found that human oocytes required at least 45 min equilibration time in glycerol with an increase in glycerol removal time (fig 1). After removal of cryoprotectant freshly produced sperm was added to media containing oocytes in an attempt at fertilisation. Fertilisation was not observed in any of the oocytes so treated. High survival rates of eight-cell mouse embryos is possible after freezing with 1,2-propanediol as cryoprotectant and an
complications.
Walker Institute, 1964 Westwood Blvd, Suite 220, Los Angeles, California 90025, USA
K. K. JAIN
Jain KK. Sutureless microvascular anastomosis using a Nd:YAG laser. J Microsurg 1980; 1: 436-39. 2. Jam KK. Sutureless end-to-side microvascular anastomosis using Nd:YAG laser. Vasc 1.
Surg 1983; 17:
240-43.
3.
Jam KK. Handbook of laser neurosurgery. Springfield, Illinois: Charles C Thomas,
4.
Jain KK. Complications of use of Nd:YAG press).
1983. laser in neurosurgery.
Neurosurgery (in
EFFECTS OF CRYOPROTECTANTS ON HUMAN OOCYTE
SiR,-The report from Australial of a pregnancy with subsequent live birth after transfer of an eight-cell human embryo, after
Fig 2-Dimensional changes of human oocytes during addition and removal of 1,2-propanediol.
633 time of only 15 min.3When we submitted human oocytes to this regimen rapid shrinkage of the blastomere was observed followed by an equally swift expansion during equilibration. After 10 min immersion in 1,2-propanediol, oocytes had recovered to 90% of their original volume (fig 2). Removal of 1,2-propanediol was equally rapid. After the addition of sperm regular cleavage was observed, and at the eight-cell stage the experiment was terminated. With the increase in demand for in-vitro fertilisation (IVF) and embryo transfer and the low success rates so far, it would seem to be more prudent to store excess oocytes for transfer in subsequent cycles, perhaps in the more favourable hormonal environment of a natural cycle. Such a technique could be beneficial in the treatment of IVF patients where several oocytes are recovered, but clearly much work is needed before a protocol for the successful storage and recovery of human oocytes can be devised.
equilibration
TABLE I-EFFECT OF INTRAPERITONEAL IMIPENEM ON GUINEAPIGS INFECTED WITH L PNEUMOPHILA
Imipenem given means
over a
period of 5 days beginning 1 day post infection. Temperatures day 4 controls (two) and 20 mglday group (three).
of six values except for
TABLE II-NUMBERS OF L PNEUMOPHILA IN GUINEAPIG LUNGS AFTER
Supported in part by the Special Trustees of the Royal Free Hospital. A. BERNARD D. A. IMOEDEMHE R. W. SHAW B. FULLER
Academic
Departments of Obstetrics Gynaecology and Surgery, Royal Free Hospital School of Medicine, London NW3 2QG 1. Trounson
A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight cell embryo. Nature 1983, 305: 707. 2. Fuller BJ, Bernard A. Successful in vitro fertilization of mouse oocytes after cryopreservation using glycerol. Cryo-Letters 1984; 5: 307-12. 3. Renard JP, Babmet C. High survival of mouse embryos after rapid freezing and thawing inside plastic straws with 1,2-propanediol as cryoprotectant. J Exp Zoo 1984; 230: 443-48.
removed and diluted in distilled water. L pneumophila was then assayed by growth on BCYE agar. L pneumophila did not multiply in tissue culture medium and 0 8% digitonin did not affect the viability of the organism over 2 h. Imipenem, although not preventing death, did marginally reduce pyrexia and prolonged the average survival time, all deaths occurring within the period of therapy (table i). Lungs from guineapigs treated with imipenem contained marginally fewer L pneumophila than those from untreated infected controls (table n). Lesions in the imipenem-treated and untreated infected control groups were identical and typical of legionnaires’ disease.7 Whereas erythromycin, an antibiotic currently recommended for treatment of legionnaires’ disease, was bactericidal against intracellular L pneumophila at concentrations approximating to the MIC (0 -05 g/ml), imipenem did not affect intracellular survival at concentrations 200 times higher than its MIC (table III). Thus imipenem had no effect on lung lesions or in preventing death in guineapigs infected with L pneumophila aerosol but it was slightly beneficial in reducing pyrexia and lung infection and in extending survival time. This was probably a result of bactericidal action on extracellular organisms. In our animal model, by contrast, erythromycin and some other antibiotics eliminated L pneumophila from the lung and prevented death.9 Imipenem may reduce bacwas
EFFICACY OF IMIPENEM IN EXPERIMENTAL LEGIONNAIRES’ DISEASE
SIR,-Imipenem has been used successfully in two patients with legionnaires’ disease,’ but this drug did not prevent the death of2 guineapigs infected intratracheally with Legionella pneumophila.2 Although imipenem has a favourably low minimum inhibitory concentration (MIC) against L pneumophila (0 - 06 J4g/ml) the main factor in estimating its efficacy against this intracellular opportunistic pathogen is thought to be its penetration into phagocytes.l,2 We have evaluated the efficacy of imipenem in guineapigs infected with a small particle aerosol containing L pneumophila3and have assessed the penetration of imipenem into alveolar phagocytes infected in vivo. L pneumophila (Corby strain, serogroup 1) was grown for 4 days at 37°C on BCYE agar4and resuspended in distilled water, inocula being standardised from the total count. Respiratory infection was achieved via a Henderson apparatus in conjunction with a three-jet Colison spray to generate bacterial aerosols containing particles <5 pm in diameter at an optimum relative humidity of65%.3 The animals studied were female Dunkin-Hartley guineapigs of category 4 health statusS weighing about 330 g. Imipenem (donated by Merck Sharp and Dohme) was given intraperitoneally as 5 or 10 mg doses every 6 h over 5 days, this dose being roughly equivalent to 4 or 8 g daily in man. Average survival times were calculated, after exclusion of survivors to the 10th day or beyond, by dividing the total number of days animals survived before dying by the total number of animals which died. To obtain infected alveolar guineapigs were killed about 6 h after aerosol infection and the lungs were washed by instillation and withdrawal of 3 x 10 ml of minimum essential medium (MEM) containing sodium bicarbonate (10 mmol/1), "hepes" buffer (20 mmol/1), L-glutamine (2 mmol/1), and heparin (5 U/ml). The washings were centrifuged at 200 g for 10 min and resuspended in fresh medium containing 5% v/v normal autologous guineapig serum. Cells were standardised at 106 /ml and 0-2 ml volumes were placed in the wells of a plastic tissue culture plate and incubated for about 3 h at 37°C in 5% CO2 in air. The cells were then washed with fresh medium (0.22 ml x 2) and incubated in 0 - 2 ml of MEM containing 5% autologous guineapig serum plus antibiotics at 37°C for 24 and 48 h in 5% CO2 in air. At 0, 24, and 48 h the monolayer of cells in each well was gently washed twice with 0 - 2 ml of fresh medium, and 0 - 2 ml 0 - 8% digitonin in distilled water was added. When the cells had disintegrated the suspension
teraemia and consequent dissemination of L pneumophila but, as our studies show, this drug does not penetrate alveolar phagocytic cells TABLE III-EFFECT OF IMIPENEM ON THE REPLICATION OF L PNEUMOPHILA WITHIN GUINEAPIG ALVEOLAR PHAGOCYTES
phagocytes
Figures in body of table denote log (viable organisms per well) and are the mean of2 values. Deviation between values was <0’3 3 log. Changmg imipenem or erythromycin at 6 or 8 hourly intervals respectively in a parallel series of wells did not alter the counts at 24 h.
,